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Team:EPF-Lausanne/Protocols/Competent cells. Protocol II
From 2011.igem.org
CaCl2 Chemical Competence (for E. coli)
Uses
TB buffer is used to make chemically competent cells by the Inoue method Preparing chemically competent cells (Inoue)
Materials
- PIPES buffer 0.5M solution pH 6.7 (sterilized, made with milli-Q pure H2O, store at -20C)
- Manganese chloride tetrahydrate
- Calcium chloride dihydrate
- Potassium chloride
- Potassium hydroxide 1M solution
- Sterile 0.22 μm bottle top filter
- vacuum source
- clean glassware (no detergent residue -- clean with 70% EtOH)
Preparation
For a 1 liter solution add:
- 250 mM potassium chloride (18.65 g)
- 15 mM calcium chloride (2.2 g)
- 10 mM PIPES (20 ml of an 0.5 M solution)
to 800 ml of DI water. Adjust pH to 6.7 with 1M KOH.
Dissolve
- 55 mM manganese chloride (10.88 g)
in 100 ml of DI water, add gradually to the solution of the remaining components. Bring solution to 1 liter. The pH of the solution will fall, which is expected.
- Do not attempt to adjust the pH. Adding base after adding manganese will precipitate a yellow/brown hydroxide.
Sterile filter with a pre-washed 0.22 μm filter and store indefinitely at 4 degrees.
